Synthesis and evaluation of 5′-modified 2′-deoxyadenosine analogues as anti-hepatitis C virus agents

Article abstract of DOI:10.1016/j.bmcl.2008.07.015

In order to study the effect of 5′-modification of 2′-deoxynucleoside on its anti-HCV activity, several analogues were synthesized and evaluated. Among the analogues, a 5′-deoxy-5′-phenacylated analogue exhibited a good anti-HCV activity with an EC₅₀ of 15.1 μM. This compound is expected to operate via a type of mechanism that does not involve a generally known 5′-O-triphosphorylation process.

Full text of DOI:10.1016/j.bmcl.2008.07.015

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4638–4641
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 50-modified 20-deoxyadenosine analogues as
anti-hepatitis C virus agents
a
a
c
a,
*
Masahiro Ikejiri a,b, , Takayuki Ohshima , Akemi Fukushima , Kunitada Shimotohno , Tokumi Maruyama
*
a Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
b Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan
c Center for Integrated Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan
a r t i c l e i n f o
a b s t r a c t
In order to study the effect of 50-modification of 20-deoxynucleoside on its anti-HCV activity, several ana-
logues were synthesized and evaluated. Among the analogues, a 50-deoxy-50-phenacylated analogue
Article history:
Received 9 June 2008
Revised 3 July 2008
Accepted 5 July 2008
Available online 10 July 2008
exhibited a good anti-HCV activity with an EC50 of 15.1 lM. This compound is expected to operate via
a type of mechanism that does not involve a generally known 50-O-triphosphorylation process.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Antiviral agent
Hepatitis C virus
HCV
Nucleoside
Hepatitis C virus (HCV)1 is a major causative agent of non-A and
non-B hepatitis. It is estimated to have infected >170 million indi-
viduals, that is, 3.5% of the world’s population. HCV infection is a
leading cause of chronic hepatitis, liver cirrhosis and hepatocellu-
lar carcinoma. Current therapy based on pegylated interferon and
ribavirin is often poorly tolerated and is effective in only 50% of pa-
tients. Therefore, the development of further effective therapeutic
agents against HCV is an urgent public health requirement.
are often hydrolyzed in cultured cells; in other words, there is a
possibility that compound 1 simply operates as a prodrug of 3.4,5
Therefore, in order to confirm the effectiveness of 50-O-masking
groups, particularly that of the benzoyl group of compound 1, we
planned the syntheses and anti-HCV evaluations of ketone ana-
logues 4 and 5, in which the 50-oxgen atom was replaced with a
methylene group to prevent the hydrolytic removal of the benzoyl
group.
In our previous study,2 we revealed that several 50-O-masked
analogues of 6-chloropurine-20-deoxyriboside, such as benzoate 1
and benzyl ether 2, exhibit an effective anti-HCV activity in a sub-
genomic replicon cell line and are more potent than the corre-
sponding unmasked analogue 3 (Fig. 1). Since it is generally
accepted that most nucleoside antivirals exhibit their potency after
being converted to the corresponding 50-triphosphates,3 the un-
masked (or phosphorylated) 50-hydroxyl group is indispensable
for the antiviral activity. Accordingly, our result that the 50-O-
masking leads to an improvement in the anti-HCV activity appears
to be inconsistent with the common understanding, interestingly.
We presume that the anti-HCV activity of certain 50-O-masked
analogues would arise from a new type of mechanism that does
not involve the 50-O-triphosphorylation process. However, there
is still room for the discussion on the 50-O-masking effect because
certain carbon–oxygen bonds, for example, the carboxylic ester
bond of compound 1 (i.e., the benzoate moiety in compound 1),
The synthesis of 4 began with readily available 30-O-TBS-20-
deoxyadenosine (6)6 (Scheme 1). First, we attempted to subject
* Corresponding authors.
E-mail address: ikejim@osaka-ohtani.ac.jp (M. Ikejiri).
Figure 1. Structures of 50-modified analogues.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.015

M. Ikejiri et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4638–4641
4639
moderate (entries 2 and 3). The use of elemental magnesium in
methanol led to a complex mixture (entry 4). In the case of 10,
chemoselective hydrogenations by Sajiki’s procedures (Pd/fi-
broin–H2 or Pd/C–Ph2S–H2)14 were ineffective, resulting in the
recovery of a large amount of the starting material (entries 5 and
6), while the 1,4-reduction with indium hydride generated in situ
15
by using PhSiH3–In(OAc)3 or Bu3SnH–InCl3 efficiently afforded
the desired product 14 in good yields (entries 7 and 8). Conse-
quently, the conditions in the case of entries 1 and 7 were em-
ployed for routine syntheses of 13 and 14, respectively, in view
of their simple experimental procedures as well as their good
yields.
Compound 13 was readily converted to 14 with a two-step se-
quence, that is, via a Weinreb amide 15, as illustrated in Scheme 2-A.
Using a Grignard reagent (PhMgBr) led to a better yield (71%) than
when phenyl lithium was used (58% yield). This two-step conver-
sion will effectively serve for the synthesis of various analogues
because the phenyl moiety of 14 can be easily replaced with other
groups by changing the type of Grignard reagent. The amino group
of 14 was substituted by a chloro group to afford 16 (40% yield),
compd
R2
yield (%)
E:Z
9
MeO–
Ph–
H–
90
60
—
—
E only
13:1
10
11
12
Me(MeO)N–
Scheme 1.
the isolated aldehyde 7 obtained via the oxidation of 6 to the fol-
lowing Wittig reaction; however, it was unsuccessful due to the
instability of 7. This issue was overcome by using a one-pot oxida-
tion–Wittig reaction with Dess–Martin periodinane (DMP) and sta-
bilized phosphorus ylide.7 Among the four types of ylides
examined (8a–d), two of them (8a and 8b) successfully afforded
the desired compounds 9 and 10 in 90% (E-isomer only) and 60%
(E:Z = 13:1) yields, respectively, while the others (8c and 8d)
yielded complex mixtures. Since the Dess–Martin oxidation is
not very suitable for the large-scale synthesis of 9 and 10 because
of the explosive nature of DMP (and also its precursor, 2-iodoxy-
benzoic acid8), several other one-pot protocols such as Moffatt
oxidation–Wittig,9 PCC–Wittig,10 TEMPO–BAIB–Wittig,11 and
TPAP–NMO–Wittig12 were examined with 6 and 8a. However,
the TLC analyses of all the attempts revealed low yields and/or
the formation of by-products.
With the thus-obtained products, the reduction of the C–C dou-
ble bond was examined (Table 1). Compound 9 was converted to
13 under standard hydrogenation conditions (Pd/C, H2, THF) with
an excellent yield although the reaction required a long reaction
time (ꢀ2 days) and comparatively large quantities of the catalyst
(50 wt.%) (entry 1). In contrast, the conjugate reduction of 9 by so-
dium borohydride–transition metal salt (e.g., NiCl2 and CuCl) sys-
tems13 furnished 13 in a short time (1–3 h), but the yield was
Table 1
Chemoselective reduction of C–C double bond
Entry
Substrate
Conditions
Product
Yield (%)
1
2
3
4
5
6
7
8
9
9
9
Pd/C, H2, THF, rt, 2 d
13
13
13
94
50
76
NiCl2, NaBH4, MeOH, 0 °C, 1 h
CuCl, NaBH4, MeOH, 0 °C, 2.5 h
Mg, MeOH, reflux, 2 h
Pd/fibroin, H2, MeOH, rt, 2 d
Pd/C, Ph2S, H2, MeOH, rt, 2 d
PhSiH3, In(OAc)3, EtOH, rt, over night
Bu3SnH, InCl3, i-PrOH ꢁ78 °C to rt, 2 h
9
Complex mix.
10
10
10
10
14
14
14
14
Tracea
31b
84
93
Scheme 2. Reagents: (a) Me(MeO)NHꢂHCl, n-BuLi, THF; (b) PhMgBr, THF;
(c) t-BuONO, Et4NCl, CCl4–CH2Cl2; (d) TBAF, AcOH, THF; (e) Et3Nꢂ3HF, THF; (f)
TAS-F, MeCN.
a
93% of 10 was recovered.
56% of 10 was recovered.
b

4640
M. Ikejiri et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4638–4641
which was subsequently treated with a mixture of tetrabutylam-
monium fluoride (TBAF) and acetic acid, giving the desired product
4 in 91% yield. A moderate yield of 16 was mainly obtained due to
the competitive elimination of its nucleobase moiety. The other de-
sired compound 5 was prepared in 92% yield by exposing 14 to tri-
ethylamine trihydrofluoride.
Since we are interested in the structure–activity relationship
(SAR) of not only the benzoyl moiety but also the methyl ester
and Weinreb amide moieties contained in the synthetic intermedi-
ates, we conducted syntheses of the corresponding analogues
17–20, as shown in Scheme 2-B. 6-Chloropurine analogues 17
and 19 were prepared from 13 and 15, respectively, under
conditions almost identical to those used in the synthesis of 4
(i.e., t-BuONO–Et4NCl and TBAF–AcOH). The conversion to 18 and
20 was effectively accomplished by the treatment of 13 and 15
with
tris(dimethylamino)sulfonium
difluorotrimethylsilicate
(TAS-F),16 while that with TBAF led to a mixture of the desired
product and certain tertabutylammonium salts that were difficult
to separate.
The synthesized nucleoside analogues mentioned above were
assayed for their ability to inhibit HCV RNA replication in a subge-
nomic replicon Huh7 cell line (LucNeo#2),17 and the result is pre-
sented in Table 2 and Figure 2. These cells contain an HCV
subgenomic replicon RNA encoding a luciferase reporter gene as
a marker. The antiviral potency of the analogues against the HCV
replicon is expressed as EC50, which was quantified by a luciferase
assay after a two-day incubation period with the corresponding
compound. In addition, the associated cytotoxicity (expressed as
CC50 in Table 2) was evaluated in a tetrazolium (XTT)-based assay
according to the manufacturer’s protocol.
Figure 2. Anti-HCV activity and cytotoxicity of 4: (A) result of luciferase assay and
XTT assay; (B) result of real-time RT-PCR.
25%, respectively. This result is almost consistent with that of the
luciferase assay with 4.
Taking these data into account, it appears that the phenacyl
group (BzCH2–) equipped at the C50 position as well as the ben-
zoyloxy group (BzO–) is effective functional group for anti-HCV
activity; this should be noteworthy because the 50-phenacyl group
is expected to operate without being converted to the correspond-
ing 50-hydroxyl group (or 50-triphosphate group). This result
strongly supports our hypothesis that the 50-O-masking group
can contribute to the anti-HCV activity not only as a unit for the
prodrug system but also as a part of the substrate. Although the de-
tailed mechanism is unclear and the biological activity is still insuf-
ficient, the antiviral potency of such 50-modified analogues is of
great interest because they are likely to operate via a pathway that
does not involve the 50-O-phosphorylation process. We hope that
the present study will contribute to developing a new class of
HCV therapeutic agents.
As shown in Table 2, the ketone analogue 4 exhibited an antivi-
ral activity against the HCV replicon with an EC50 of 15.1
1), which is nearly comparable to that of benzoate analogue 1 (en-
try 7). The cytotoxicity of 4 was somewhat high (CC50: 76.3 M),
but was not high enough to exert an influence on the EC50 value
because the cytotoxicity at 15 M was considerably low (ca. 0–
lM (entry
l
l
2%) (Fig. 2A). Thus, the decrease in the luciferase activity with 4 re-
sults from its anti-HCV activity, not its cytotoxicity. Interestingly,
compounds 17 and 19 also exhibited anti-HCV activities (entries
3 and 5, respectively). In contrast, the 6-amino analogues 5, 18,
and 20 did not exhibit any significant anti-HCV activity (entries
2, 4, and 6).18
To confirm the anti-HCV potency of compound 4, subgenomic
replicon RNA levels were quantified by real-time RT-PCR analysis
Acknowledgments
(Fig. 2B). Exposing the replicon cells to 12.5 and 25 lM of 4 re-
duced the replicon RNA amount up to approximately 60% and
This research was partly supported by a Grant-in-Aid for Young
Scientists (B) (20790106) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and by a Grant-in-Aid
from Mitsubishi Chemical Corporation Fund.
Table 2
Inhibitory potency (EC50) and cytotoxicity (CC50) of the synthesized analogues in HCV
replicon assay
Supplementary data
Supplementary data associated with this article (experimental
details and spectroscopic data of new compounds 4–6, 9, 10, 13–
21) can be found, in the online version, at doi:10.1016/j.bmcl.
2008.07.015.
a
a
Entry
Compound
R
X
B
EC50
(lM)
CC50 (lM)
References
1
2
3
4
5
6
7
4
5
Ph
Ph
MeO
MeO
Me(MeO)N
Me(MeO)N
Ph
CH2
CH2
CH2
CH2
CH2
CH2
O
CP
A
CP
A
CP
A
CP
15.1 0.4
>200
32.9 1.6
>200
76.3 5.2
—
>200
—
1. Recent reviews: (a) Gordon, C. P.; Keller, P. A. J. Med. Chem. 2005, 48, 1; (b) De
Francesco, R.; Migliaccio, G. Nature 2005, 436, 953; (c) De Clercq, E. Nat. Rev.
Drug Discov. 2007, 6, 1001.
2. Ikejiri, M.; Ohshima, T.; Kato, K.; Toyama, M.; Murata, T.; Shimotohno, K.;
Maruyama, T. Bioorg. Med. Chem. 2007, 15, 6882.
17
18
19
20
1
40.4 1.4
>200
>200
—
6.1b
111b
3. Arimilli, M. N.; Dougherty, J. P.; Cundy, K. C.; Bischofberger, N.. In Advances
in Antiviral Drug Design; De Clercq, E., Ed.; Jai Press Inc.: Stamford
a
EC50: 50% effective concentration; CC50: 50% cytotoxic concentration.
Extracts obtained from our previous study (Ref. 2).
Connecticut, 1999; Vol. 3, pp 69–91. and also see Refs. 1a and
references therein.
c and
b

M. Ikejiri et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4638–4641
4641
4. Several 50-O-acyl nucleoside analogues have been reported as prodrugs of the
corresponding deacylated analogues Parang, K.; Wiebe, L. I.; Knaus, E. E. Curr.
Med. Chem. 2000, 7, 995.
13. (a) Narisada, M.; Horibe, I.; Watanabe, F.; Takeda, K. J. Org. Chem. 1989,
54, 5308; (b) Satoh, T.; Nanba, K.; Suzuki, S. Chem. Pharm. Bull. 1971,
19, 817.
5. Not only compound 1 but also 2 might operate as a prodrug of 3 since
O-dealkylated metabolism is caused in some cases. Silverman, R. B. The
Organic Chemistry of Drug Design and Drug Action, Second Edition; Elsevier,
2004, Chapter 7.
6. Somu, R. V.; Wilson, D. J.; Bennett, E. M.; Boshoff, H. I.; Celia, L.; Beck, B. J.;
Barry, C. E., III; Aldrich, C. C. J. Med. Chem. 2006, 49, 7623.
14. (a) Ikawa, T.; Sajiki, H.; Hirota, K. Tetrahedron 2005, 61, 2217; (b) Mori, A.;
Mizusaki, T.; Miyakawa, Y.; Ohashi, E.; Haga, T.; Maegawa, T.; Monguchi, Y.;
Sajiki, H. Tetrahedron 2006, 62, 11925.
15. (a) Miura, K.; Yamada, Y.; Tomita, M.; Hosomi, A. Synlett 2004, 1985; (b) Inoue,
K.; Ishida, T.; Shibata, I.; Baba, A. Adv. Synth. Catal. 2002, 344, 283.
16. Kang, S. B.; De Clercq, E.; Lakshman, M. K. J. Org. Chem. 2007, 72, 5724.
17. (a) Watashi, K.; Hijikata, M.; Hosaka, M.; Yamaji, M.; Shimotohno, K.
Hepatology 2003, 38, 1282; (b) Murata, T.; Hijikata, M.; Shimotohno, K.
Virology 2005, 340, 105; (c) Goto, K.; Watashi, K.; Murata, T.; Hishiki, T.;
Hijikata, M.; Shimotohno, K. Biochem. Biophys. Res. Commun. 2006, 343, 879.
And also see Ref. 2.
7. Barrett, A. G. M.; Hamprecht, D.; Ohkubo, M. J. Org. Chem. 1997, 62, 9376.
8.
A
one-pot oxidation–Wittig reaction with 2-iodoxybenzoic acid is also
reported Crich, D.; Mo, X.-S. Synlett 1999, 67.
9. Rapp, M.; Haubrich, T. A.; Perrault, J.; Mackey, Z. B.; McKerrow, J. H.; Chiang, P.
K.; Wnuk, S. F. J. Med. Chem. 2006, 49, 2096.
10. Bressette, A. R.; Glover, L. C., IV Synlett 2004, 738.
11. Vatèle, J.-M. Tetrahedron Lett. 2006, 47, 715.
12. MacCoss, R. N.; Balskus, E. P.; Ley, S. V. Tetrahedron Lett. 2003, 44, 7779.
18. A similar SAR trend was observed in our previous study. Ikejiri, M.; Saijo, M.;
Morikawa, S.; Fukushi, S.; Mizutani, T.; Kurane, I.; Maruyama, T. Bioorg. Med.
Chem. Lett. 2007, 17, 2470.